Biotin Derivatives

ABSTRACT

Biotin derivatives, methods of using the biotin derivatives and kits comprising the biotin derivatives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is Divisional of U.S. application Ser. No. 13/881,518,filed Sep. 26, 2013, which is a U.S. National Stage Application of PCTApplication Serial No. PCT/US2011/058455, filed Oct. 28, 2011, whichclaims the benefit of priority to U.S. Application Nos. 61/408,210,filed Oct. 29, 2010, 61/495,070, filed Jun. 9, 2011, 61/501,500, filedJun. 27, 2011, and 61/510,949, filed Jul. 22, 2011, which disclosuresare herein incorporated by reference in their entirety.

BACKGROUND

The interaction between biotin and avidin or streptavidin is consideredto be practically irreversible. Biotin has a affinity constant, orK_(D), for avidin of about 10⁻¹⁵ M. While the interaction provides anexcellent means for capturing biotin-containing entities, releasingthose entities once captured requires very harsh conditions, such as astrong acid, detergent, and/or high temperature. Such conditionsinclude, for example, boiling in high salt; formamide and EDTA heated to94° C.; 6 M guanidine, pH 1.5; and heating to at least 65° C. in thepresence of salt, SDS, and EDTA. Such conditions are generally notsuitable for purification of proteins or viable cells or viruses.

SUMMARY

The inventors have developed biotin derivatives that have reducedaffinity for streptavidin. Thus, in some embodiments, the biotinderivatives can be separated from streptavidin under less harshconditions than are required for biotin. Accordingly, in someembodiments, the biotin derivatives are better suited for applicationsin which it would be desirable to dissociate the biotin derivative fromstreptavidin under less harsh conditions, such as to maintain thestructural integrity and/or viability of the moiety attached to thebiotin derivative.

In some embodiments, biotin derivatives are provided. In someembodiments, a biotin derivative is provided that has the formula:

wherein:

X is S or O;

R₁ is selected from H and a derivative group;

R₂ is selected from H and a derivative group;

Y is O or absent;

L is absent or is a linker;

R₃ is selected from —OR₄, —COOR₄, a reactive group, a protein, apeptide, an amino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle;

R₄ is selected from H and a derivative group;

wherein at least one of R₁ and R₂ is H;

wherein if X is O, at least one of R₁ and R₂ is not H, or Y is O.

In some embodiments, if R₁ is H, R₂ is a derivative group and if R₂ isH, R₁ is a derivative group. In some embodiments, R₁ is a derivativegroup. In some embodiments, R₁ comprises 1 to 8 carbon atoms, 1 to 6carbon atoms, or 1 to 3 carbon atoms. In some embodiments, R₂ is aderivative group. In some embodiments, R₂ comprises 1 to 8 carbon atoms,1 to 6 carbon atoms, or 1 to 3 carbon atoms. In some embodiments, X isO. In some embodiments, X is S. In some embodiments, Y is absent. Insome embodiments, Y is O.

In some embodiments, a biotin derivative is provided that has theformula:

wherein:

X is S or O;

R₁ is selected from H and a derivative group;

R₂ is selected from H and a derivative group;

L is absent or is a linker;

R₃ is selected from —OR₄ or —COOR₄, a reactive group, a protein, apeptide, an amino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle;

R₄ is selected from H and a derivative group;

wherein at least one of R₁ and R₂ is H.

In some embodiments, if R₁ is H, R₂ is a derivative group, and if R₂ isH, R₁ is a derivative group. In some embodiments, R₁ is a derivativegroup. In some embodiments, R₁ comprises 1 to 8 carbon atoms, 1 to 6carbon atoms, or 1 to 3 carbon atoms. In some embodiments, R₂ is aderivative group. In some embodiments, R₂ comprises 1 to 8 carbon atoms,1 to 6 carbon atoms, or 1 to 3 carbon atoms. In some embodiments, X isO. In some embodiments, X is S. In some embodiments, R₁ and R₂ are eachH.

In some embodiments of the biotin derivatives of Formulae I and II, L isabsent. In some embodiments, L is a linker. In some embodiments, L isselected from a polyethylene glycol linker and an oligopeptide linker.In some embodiments, the linker comprises a detectable moiety. In someembodiments, the detectable moiety is selected from achromophore-containing moiety, a fluorescent moiety, an affinity tag, achemiluminescent moiety, an enzyme, and an antibody. In someembodiments, the detectable moiety is selected from a chromophore, afluorescent dye, a fluorescent protein, a phosphorescent dye, a tandemdye, a particle, a nanocrystal, a hapten, an enzyme and a moleculecomprising an atom which is a radioisotope. In some embodiments, thedetectable moiety is selected from a fluorescent dye, a fluorescentprotein, a nanocrystal, an enzyme, and a molecule comprising an atomwhich is a radioisotope. In some embodiments, L-R₃ has the structure:

In some embodiments, R₅ is H or a derivative group. In some embodiments,n is an integer from 0 to 20.

In some embodiments of Formulae I and II, R₃ is —OR₄. In someembodiments, R₃ is —COOR₄. In some embodiments, R₄ is H. In someembodiments, R₄ comprises 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1to 3 carbon atoms. In some embodiments, R₄ is CH₃. In some embodiments,R₃ is a reactive group. In some embodiments, the reactive group isselected from isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl,a carboxylate, a halo acetyl, hydrazide, a succinimidyl ester, a4-sulfonyl-3,5-dichlorophenol ester, a maleimide, an iodoacetamide; anazide, and an alkyne, when L is a linker; or is selected from hydroxyl,hydrazinyl, N-hydroxysuccinimidyl, and 4-sulfonyl-3,5-dichlorophenolwhen L is absent. In some embodiments, R₃ is

when L is a linker; or

when is L is absent.In some embodiments, R₃ is selected from a polypeptide, a peptide, anamino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle.

In some embodiments, a biotin derivative has an affinity forstreptavidin of between 1×10⁻¹³ M and 1×10⁻⁸ M.

In some embodiments, compositions comprising a biotin derivative and asolvent are provided. In some embodiments, the solvent is an aqueoussolvent.

In some embodiments, methods of immobilizing a biotin derivative to asolid support are provided. In some embodiments, the method comprisescontacting a solid support with a biotin derivative, wherein the solidsupport comprises a biotin-binding moiety. In some embodiments, thebiotin derivative has an affinity for the biotin-binding moiety that isless than the affinity of biotin for the biotin-binding moiety. In someembodiments, the method further comprises contacting the immobilizedbiotin derivative with a molecule that has an affinity for thebiotin-binding moiety that is greater than the affinity of the biotinderivative for the biotin-binding moiety. In some embodiments, themolecule is biotin. In some embodiments, the molecule is a biotinmultimers. In some embodiments, the molecule is a biotin dimer or abiotin trimer. In some embodiments, the biotin-binding moiety isselected from avidin and streptavidin. In some embodiments, the solidsupport is selected from surfaces of polymer, glass, ceramic, silicone,metal, cellulose, and gel. In some embodiments, the solid support isselected from a microplate, a microarray chip, and a microparticle. Insome embodiments, the biotin derivative comprises a group selected froma protein, a peptide, an amino acid, a dextran, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,and a cell.

In some embodiments, kits are provided. In some embodiments, a kitcomprises a biotin derivative and/or a composition comprising a biotinderivative.

In some embodiments, methods of making biotin derivatives are provided.

In some embodiments, a method comprises synthesizing a biotin derivativehaving the structure:

In some embodiments, the method comprises the step of reacting biotinmethyl ester with Lawesson's reagent.

In some embodiments, a method comprises synthesizing a biotin derivativehaving the structure:

In some embodiments, the method comprises the steps of (a) reactingbiotin methyl ester with Lawesson's reagent; and (b) oxidizing the ringsulfur atom with hydrogen peroxide.

In some embodiments, a method comprises synthesizing a biotin derivativehaving the structure:

wherein R₁ is a derivative group.

In some embodiments, the method comprises the step of reacting biotinmethyl ester with a secondary amine-reactive derivative group. In someembodiments, the secondary amine-reactive derivative group is selectedfrom an iodoalkyl, a bromoalkyl, a chloroalkyl, and an alkylchloroformate.

In some embodiments, a method comprises synthesizing a biotin derivativehaving the structure:

wherein R₂ is a derivative group.

In some embodiments, the method comprises the steps of (a) reactingbiotin methyl ester with a secondary amine-reactive protecting group tomake a biotin methyl ester comprising a protecting group on a ringnitrogen; (b) reacting the product of (a) with a secondaryamine-reactive derivative group; and (c) removing the protecting group.In some embodiments, the secondary amine-reactive protecting group isselected from dimethoxytrityl chloride (DMTr-Cl), trityl chloride(Trt-Cl), benzyl chloroformate (Cbz-Cl), and allyl chloroformate(Aloc-Cl). In some embodiments, the secondary amine-reactive derivativegroup is selected from an iodoalkyl, a bromoalkyl, a chloroalkyl, and analkyl chloroformate.

In some embodiments, a method comprises synthesizing a biotin derivativehaving the formula:

wherein:

X is S or O;

R₁ is selected from H and a derivative group;

R₂ is selected from H and a derivative group;

Y is O or absent;

L is absent or is a linker;

R₃ is selected from —OR₄, —COOR₄, a reactive group, a protein, apeptide, an amino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle;

R₄ is selected from H and a derivative group;

wherein at least one of R₁ and R₂ is H;

wherein if X is O, at least one of R₁ and R₂ is not H, or Y is O.

In some embodiments, the method comprises the step of synthesizing abiotin derivative selected from:

In some embodiments, a method comprises synthesizing a biotin derivativehaving the structure:

wherein R₁ is a derivative group.

In some embodiments, the method comprises the step of reactingdesthiobiotin methyl ester with a secondary amine-reactive derivativegroup. In some embodiments, the secondary amine-reactive derivativegroup is selected from an iodoalkyl, a bromoalkyl, a chloroalkyl, and analkyl chloroformate.

In some embodiments, a method comprises synthesizing a biotin derivativehaving the structure:

wherein R₂ is a derivative group.

In some embodiments, the method comprises the steps of (a) reactingdesthiobiotin methyl ester with a secondary amine-reactive protectinggroup; and (b) reacting the product of (a) with a secondaryamine-reactive derivative group. In some embodiments, the secondaryamine-reactive protecting group is selected from dimethoxytritylchloride (DMTr-Cl), trityl chloride (Trt-Cl), benzyl chloroformate(Cbz-Cl), allyl chloroformate (Aloc-Cl). In some embodiments, thesecondary amine-reactive derivative group is selected from an iodoalkyl,a bromoalkyl, a chloroalkyl, and an alkyl chloroformate.

In some embodiments, a method comprises synthesizing a biotin derivativehaving the structure:

In some embodiments, the method comprises reacting desthiobiotin methylester with Lawesson's reagent.

In some embodiments, a method comprises synthesizing a biotin derivativehaving the formula:

wherein:

X is S or O;

R₁ is selected from H and a derivative group;

R₂ is selected from H and a derivative group;

L is absent or is a linker;

R₃ is selected from —OR₄, —COOR₄, a reactive group, a protein, apeptide, an amino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle;

R₄ is selected from H and a derivative group;

wherein at least one of R₁ and R₂ is H.

In some embodiments, the method comprises the step of synthesizing abiotin derivative selected from:

In some embodiments, a method comprises the step of converting acompound selected from the group consisting of:

to form the biotin derivative.In some embodiments, the biotin derivative made by this method has theformula:

wherein:

X is S or O;

R₁ is selected from H and a derivative group;

R₂ is selected from H and a derivative group;

Y is O or absent;

L is absent or is a linker;

R₃ is selected from —OR₄, —COOR₄, a reactive group, a protein, apeptide, an amino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle;

R₄ is selected from H and a derivative group;

wherein at least one of R₁ and R₂ is H;

wherein if X is O, at least one of R₁ and R₂ is not H, or Y is O.

In some embodiments, the biotin derivative made by this method has theformula:

wherein:

X is S or O;

R₁ is selected from H and a derivative group;

R₂ is selected from H and a derivative group;

L is absent or is a linker;

R₃ is selected from —OR₄, —COOR₄, a protein, a peptide, an amino acid, adextran, a monosaccharide, a disaccharide, a trisaccharide, anoligosaccharide, a polysaccharide, a hormone, a lipopolysaccharide, anucleotide, an oligonucleotide, a small molecule, a cell, a microplate,and a microparticle;

R₄ is selected from H and a derivative group;

wherein at least one of R₁ and R₂ is H.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show purification of protein labeled with biotinderivative 21 from a cell lysate mixture. FIG. 1A shows the fluorescencelabeled protein distribution before and after streptavidin-coatedagarose capture (left tube and center tube, respectively), and afterrelease from the streptavidin coated agarose (right tube). FIG. 1B showsseparation of the proteins in the cell lysate mixture (lane 1), in thesupernatant following capture of the biotin derivative labeled protein(lane 2), in the wash (lane 3), and the purified protein released fromthe streptavidin coated agarose (lane 3).

FIG. 2 shows isolation of CD3+ T cells from a mononuclear cell poolusing an anti-CD3 antibody linked to a biotin derivative 17. Step Ashows the CD3+ T cells from a mononuclear cell pool, bound to anti-CD3antibody labeled with biotin derivative 17, the complexes of which arebound to M280 Strepavidin Dynabeads. Step B shows the isolated CD3+ Tcells, which have been disassociated from the M280 StrepavidinDynabeads. Step C shows the M280 Streptavidin Dynabeads afterdissociation of CD3+ T cell/anti-CD3 antibody complexes. (The sizes ofthe disassociated CD3+ T cell complexes shown in Step B and thedisassociated M280 Strepavidin Dynabeads shown in Step C are not to thesame scale as FIG. 1A.)

FIG. 3 shows the association and dissociation curves between N3′-ethylbiotin and streptavidin on ForteBio system.

FIG. 4 shows the fluoresence microscopy of HeLa cells labeled withN3′-ethyl biotin alkyne 118. Cells incubated with Ac₄MaNAz supplementedmedia (top) or unsupplemented media (bottom) were labeled N3′-ethylbiotin alkyne 118 in the presence of Cu(I)/THPTA as catalyst.Fluorescence signal from AF488 (left), the nuclear stain, Hoechst 33342,(center) and the merged images (right) are shown.

FIG. 5 shows the fluoresence microscopy of HeLa cells labeled withN3′-ethyl biotin alkyne 118. Cells incubated with Ac4GlcNAz, Ac4GalNAz,or Ac₄MaNAz supplemented media (top) were labeled N3′-ethyl biotinalkyne 118 in the presence of Cu(I)/THPTA as catalyst (rows 1-3) or inabsence Cu(I) (rows 4-6). Cells not incubated with Ac4GlcNAz, Ac4GalNAz,or Ac₄MaNAz were treated with N3′-ethyl biotin alkyne 118 in thepresence of Cu(I)/THPTA as catalyst as a control (row 7). Fluorescencesignal from AF488 (left), the nuclear stain, Hoechst 33342, (center) andthe merged images (right) are shown.

FIG. 6 shows the isolation of glycoproteins using click chemistry. Celllysate was incubated with N3′-ethyl biotin alkyne 118 or biotin alkynein the presence of Cu(I)/THPTA, captured by streptavidin agarose,washed, and eluted with either 10 mM HCl or 1% SDS in H₂O at 95° C.,respectively. The purified fractions were analyzed by a reducing 4-12%SDS-PAGE gel and stained with SYPRO-Ruby.

FIG. 7A shows an illustration of cell separation work-flow; FIG. 7Bshows cell imaging at different separation stages. Scale bar, 10 am; andFIG. 7C shows flow histograms of cells based on three different cellsurface markers (CD3, CD4, and CD8) at pre-isolation, after depletion,and after release, respectively.

FIG. 8 shows flow histograms of cells based on dual cell surface markers(CD3 and CD4) at pre-isolation, after depletion, and after release.Population of lymphocytes, monocytes, and granulocytes shown on top.Subpopulation of lymphocytes based on CD3 and CD4 cell surface markersshown at bottom.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Exemplary techniques used in connection with recombinant DNA,oligonucleotide synthesis, tissue culture, enzymatic reactions, andpurification are known in the art. Many such techniques and proceduresare described, e.g., in Sambrook et al. Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), among other places. In addition, exemplarytechniques for chemical syntheses are also known in the art.

In this application, the use of “or” means “and/or” unless statedotherwise. In the context of a multiple dependent claim, the use of “or”refers back to more than one preceding independent or dependent claim inthe alternative only. Also, terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit unless specificallystated otherwise.

Biotin Derivatives

The present invention provides biotin derivatives that are useful forapplications in which it would be desirable to dissociate the biotinderivative from, for example, strepavidin under less harsh conditions,or where it would be desirable not to have such dissociation to occur.As used herein, the term “biotin derivative” includes, but is notlimited to, derivatives of biotin or desthiobiotin which have adissociation constant that is either less than or greater than thedissociation constant of biotin or desthiobiotin for either avidin orstrepavidin.

In some embodiments, a biotin derivative is provided that has theformula:

In some embodiments, X is S or O. In some embodiments, R₁ is selectedfrom H and a derivative group. In some embodiments, R₂ is selected fromH and a derivative group. In some embodiments, Y is O or absent. In someembodiments, L is absent or is a linker. In some embodiments, at leastone of R₁ and R₂ is H. In some embodiments, if X is O, at least one orR₁ and R₂ is not H. In some embodiments, if X is O, Y is O. In someembodiments, if R₁ is H, R₂ is a derivative group. In some embodiments,if R₂ is H, R₁ is a derivative group. In some embodiments, R₃ isselected from —RO₄, —COOR₄, a reactive group, a protein, a peptide, anamino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle.

In some embodiments, a biotin derivative is provided that has theformula:

In some embodiments, X is S or O. In some embodiments, R₁ is selectedfrom H and a derivative group. In some embodiments, R₂ is selected fromH and a derivative group. In some embodiments, L is absent or is alinker. In some embodiments, if X is S, at least one of R₁ and R₂ is H.In some embodiments, if X is O, at least one of R₁ and R₂ is not H. Insome embodiments, if R₁ is H, R₂ is a derivative group. In someembodiments, if R₂ is H, R₁ is a derivative group. In some embodiments,R₃ is selected from —RO₄, —COOR₄, a reactive group, a protein, apeptide, an amino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle.

The term “derivative group” as used herein refers to a substituted orunsubstituted straight chain, branched chain, cyclic, or a combinationthereof, carbon radical that may be fully saturated, mono-unsaturated,or poly-unsaturated, and which comprises 1 to 12 carbon atoms (i.e., C₁to C₁₂). Exemplary saturated groups include, but are not limited to,alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, and homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like. Anunsaturated group is a group having one or more double bonds, such asalkenyl groups, or triple bonds, such as alkynyl groups. Examples ofunsaturated derivative groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher (i.e., having more carbon atoms) homologs and isomers.

The term derivative group includes groups that comprise one or moreheteroatom substituents selected from O, N, Si, P, and S. Exemplary suchderivative groups include straight chain, branched chain, cyclic, or acombination thereof, carbon-containing radicals, which comprise thestated number of carbon atoms and at least one heteroatom selected fromO, N, Si, P, and S, and wherein the nitrogen, phosphorous and/or sulfuratoms may be optionally oxidized, and a nitrogen heteroatom may beoptionally be quaternized. In some embodiments, one or more heteroatomsmay be placed at interior positions of the derivative group, or at aposition at which the derivative group is attached to the remainder of amolecule. Thus, derivative groups include, but are not limited to,alkoxy groups, alkylamino groups, and alkylthio groups.

The term derivative group also includes groups comprising carbon atomssubstituted with one or more halogens in place of one or more hydrogens.Exemplary halogens that may be present in such derivative groups includeF, Br, Cl, and I.

In some embodiments, a derivative group comprises 1 to 11 carbon atoms(i.e., C₁ to C₁₁), 1 to 10 carbon atoms (i.e., C₁ to C₁₀), 1 to 9 carbonatoms (i.e., C₁ to C₉), 1 to 8 carbon atoms (i.e., C₁ to C₈), 1 to 7carbon atoms (i.e., C₁ to C₇), 1 to 6 carbon atoms (i.e., C₁ to C₆), 1to 5 carbon atoms (i.e., C₁ to C₅), 1 to 4 carbon atoms (C₁ to C₄), 1 to3 carbon atoms (C₁ to C₃), or 1 to 2 carbon atoms (C₁ to C₂). In someembodiments, derivative group is a saturated or unsaturated straightchain or branched chain carbon radical.

The term “linker” as used herein refers to a chemical moiety that linksa biotin derivative of any one of Formulae I to VI to another molecule,such as a reactive group, a protein, a peptide, an amino acid, adextran, a monosaccharide, a disaccharide, a trisaccharide, anoligosaccharide, a polysaccharide, a hormone, a lipopolysaccharide, amolecule on the surface of a cell, a nucleotide, an oligonucleotide, asmall molecule, a molecule on the surface of a microplate, and amolecule on the surface of a microparticle.

In some embodiments, a linker is a polymer or a biopolymer. Nonlimitingexemplary linkers include polyethylene glycol linkers and oligopeptidelinkers (also referred to simply as peptide linkers). Many linkers areknown in the art. One skilled in the art can select a suitable linkeraccording to the intended application.

In some embodiments, a linker comprises a detectable moiety.

The term “detectable moiety” as used herein refers to a moiety thatfacilitates detection of a molecule, and would include any moietydetectable by any means known in the art. Nonlimiting exemplarydetectable moieties include chromophore-containing moieties, fluorescentmoieties, affinity tags, chemiluminescent moieties, enzymes, antibodies,and a molecule comprising an atom which is a radioisotope. Manydetectable moieties are known in the art which include, but are notlimited to, a chromophore, a fluorescent dye, a fluorescent protein, aphosphorescent dye, a tandem dye, a particle, a nanocrystal, a hapten,an enzyme, a molecule comprising an atom which is a radioisotope or anatom which is a radioisotope. Numerous fluorescent dyes are known tothose skilled in the art and include, but are not limited to, coumarin,cyanine, benzofuran, a quinoline, a quinazolinone, an indole, abenzazole, a borapolyazaindacene and xanthenes including fluoroscein,rhodamine and rhodol. The presence of a detectable moiety is, directlyor indirectly, detectable. For example, radiolabels that can be measuredwith radiation-counting devices; pigments, dyes or other chromogens thatcan be visually observed or measured with a spectrophotometer; spinlabels that can be measured with a spin label analyzer; and fluorescentmoieties (fluorophores), where the output signal is generated by theexcitation of a suitable molecular adduct and that can be visualized byexcitation with light that is absorbed by the dye or can be measuredwith standard fluorometers or imaging systems. One skilled in the artcan select a suitable detectable moiety according to the intended use.

The term “reactive group” as used herein refers to a chemical moietythat is part of a first molecule that is capable of reacting with a“complementary group” that is part of a second molecule to form acovalent bond between the first molecule and the second molecule.Complementary group may also be called “complementary reactive group”.In some embodiments, a reactive group is an electrophilic group and acomplementary group is a nucleophilic group. In some embodiments, areactive group is a nucleophilic group and a complementary group is anelectrophilic group. In some embodiments, the reactive group is an azideand the complementary group is an alkyne. In some embodiments thereactive group is an alkyne and the complementary group is an azide. Theterm, “alkyne”, includes, but is not limited to, a moiety comprising aterminal carbon-carbon triple bond, such as, for example, acetylene. Theterm also includes, but is not limited to, a moiety comprising anactivated carbon-carbon triple bond, such as for example, cyclooctynesand difluorcyclooctynes, dibenzocyclooctynes, andaza-dibenzocyclooctynes. In some embodiments, a reactive group is partof a molecule selected from Formulae I to VI. In some embodiments, acomplementary group is part of a second molecule selected from aprotein, a peptide, an amino acid, a dextran, a monosaccharide, adisaccharide, a trisaccharide, an oligosaccharide, a polysaccharide, ahormone, a lipopolysaccharide, a molecule on the surface of a cell, anucleotide, an oligonucleotide, a small molecule, a molecule on thesurface of a microplate, and a molecule on the surface of amicroparticle. In some embodiments, a complementary group is an amine.

Nonlimiting exemplary reactive groups include isothiocyanate, sulfonylchloride, 4,6-dichlorotriazinyl, carboxylates, halo acetyls (such as anacid chloride), hydrazides, succinimidyl esters (such asN-hydroxysuccinimidyl (NHS) ester), phenol esters (such as4-sulfonyl-3,5-dichlorophenol ester), maleimides, an azide, an alkyne,and iodoacetamide, when L is a linker; or hydroxyl, hydrazinyl,N-hydroxysuccinimidyl, and 4-sulfonyl-3,5-dichlorophenol when L isabsent. Nonlimiting exemplary reactive groups also include, but are notlimited to, olefins, acetylenes, alcohols, phenols, ethers, oxides,halides, aldehydes, ketones, esters, amines, amides, cyanates,isocyanates, thiocyanates, isothiocyanates, hydrazines, hydrazones,diazo groups, diazonium groups, nitro groups, nitriles, mercaptans,sulfides, disulfides, sulfoxides, sulfones, sulfonic acid groups,sulfinic acid groups, acetals, ketals, anhydrides, sulfates, sulfenicacid groups, isonitriles, amidines, imides, imidates, nitrones,hydroxylamines, oximes, hydroxamic acid groups, thiohydroxamic acidgroups, allenes, ortho esters, sulfites, enamines, ynamines, ureas,pseudoureas, semicarbazides, carbodiimides, carbamates, imines, azides,alkyne, azo groups, azoxy groups, and nitroso groups.

In some embodiments, a biotin derivative is provided that has theformula:

In some embodiments, R₁ is a derivative group. In some embodiments, L isabsent or is a linker. In some embodiments, R₃ is selected from —RO₄,—COOR₄, a reactive group, a protein, a peptide, an amino acid, adextran, a monosaccharide, a disaccharide, a trisaccharide, anoligosaccharide, a polysaccharide, a hormone, a lipopolysaccharide, anucleotide, an oligonucleotide, a small molecule, a cell, a microplate,and a microparticle.

In some embodiments, a biotin derivative is provided that has theformula:

In some embodiments, R₂ is a derivative group. In some embodiments, L isabsent or is a linker. In some embodiments, R₃ is selected from —RO₄,—COOR₄, a reactive group, a protein, a peptide, an amino acid, adextran, a monosaccharide, a disaccharide, a trisaccharide, anoligosaccharide, a polysaccharide, a hormone, a lipopolysaccharide, anucleotide, an oligonucleotide, a small molecule, a cell, a microplate,and a microparticle.

In some embodiments, a biotin derivative is provided that has theformula:

In some embodiments, R₁ is a derivative group. In some embodiments, L isabsent or is a linker. In some embodiments, R₃ is selected from —RO₄,—COOR₄, a reactive group, a protein, a peptide, an amino acid, adextran, a monosaccharide, a disaccharide, a trisaccharide, anoligosaccharide, a polysaccharide, a hormone, a lipopolysaccharide, anucleotide, an oligonucleotide, a small molecule, a cell, a microplate,and a microparticle.

In some embodiments, a biotin derivative is provided that has theformula:

In some embodiments, R₂ is a derivative group. In some embodiments, L isabsent or is a linker. In some embodiments, R₃ is selected from —RO₄,—COOR₄, a reactive group, a protein, a peptide, an amino acid, adextran, a monosaccharide, a disaccharide, a trisaccharide, anoligosaccharide, a polysaccharide, a hormone, a lipopolysaccharide, anucleotide, an oligonucleotide, a small molecule, a cell, a microplate,and a microparticle.

Certain nonlimiting exemplary biotin derivatives are shown in Tables 1,2, 3, and 4. Table 1 shows certain nonlimiting exemplary biotinderivative structures in which L and R₃ are not specified. As notedabove, L may be absent or may be a linker. Many different linkers and/orR₃ groups may be used in the structures in Table 1. One skilled in theart can select a suitable linker (or select the absence of a linker) anda suitable R₃ group according to the intended application.

Table 2 shows certain nonlimiting exemplary biotin derivative structuresin which the R₃ group is not specified. One skilled in the art canselect a suitable R₃ group according to the intended application.Certain linkers are represented in the structures in Table 2. Thoselinkers are in no way exhaustive of the possible linkers that could beused, for example, in the structures in Table 1, or in Formulae I to VI.

Table 3 shows certain nonlimiting exemplary biotin derivatives. Each ofthose biotin derivatives comprises the reactive groupN-hydroxysuccinimidyl (NHS) ester as R₃. While those structures areshown with NHS, that representation is in no way exhaustive of thepossible reactive groups, or R₃ groups, that could be used, for example,in the structures in Table 1, or in Formulae I to VI.

Table 4 shows certain nonlimiting exemplary biotin derivatives. Each ofthose structures comprises an ester group (—COOR₄), which may be acarboxylic acid when R₄ is H.

TABLE 1 Nonlimiting exemplary biotin derivative structures CompoundStructure 29

30

31

32

33

34

35

36

37

38

TABLE 2 Nonlimiting exemplary biotin derivative structures 39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

111

112

54

55

56

57

58

59

60

61

TABLE 3 Nonlimiting exemplary biotin derivative structures  1

 2

 3

 4

 5

113 

114 

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

118 

119 

TABLE 4 Nonlimiting exemplary biotin derivative structures 88

89

90

91

92

93

115

116

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

In some embodiments, R₃ is selected from a protein, a peptide, an aminoacid, a dextran, a monosaccharide, a disaccharide, a trisaccharide, anoligosaccharide, a polysaccharide, a hormone, a lipopolysaccharide, anucleotide, an oligonucleotide, a small molecule, a cell, a microplate,and a microparticle. In some embodiments, a protein R₃ group is anantibody. Such antibodies may be of any species, class, or subtype, andinclude chimeric antibodies, humanized antibodies, antibody fragments,such as Fab, Fab′, F(ab′)₂, Fv, scFv, single domain antibodies (sdAbs),etc.

In some embodiments, R₃ is —OR₄, wherein R₄ is selected from H and aderivative group. In some embodiments, R₄ comprises 1 to 8 carbon atoms,1 to 6 carbon atoms, or 1 to 3 carbon atoms. In some embodiments, R₄ ismethyl (CH₃). In some embodiments, L is absent and R₃ is —OR₄, whereinR₄ is defined as above. See nonlimiting exemplary structures in Table 4.

Exemplary Methods of Synthesizing Biotin Derivatives

In some embodiments, methods of synthesizing biotin derivatives ofFormulae I to VI are provided. Nonlimiting exemplary methods ofsynthesizing biotin derivatives of Formulae I to VI are describedherein, e.g., in Examples 1 to 11.

In some embodiments, a method of synthesizing biotin derivative 67 isprovided.

In some embodiments, the method comprises the step of reacting biotinmethyl ester with Lawesson's reagent, which has the structure:

In some embodiments, the reaction is carried out in an organic solvent,such as xylene.

In some embodiments, the ring sulfur atom in biotin derivative 67 isoxidized to sulfone using, e.g., hydrogen peroxide ormeta-chloroperoxybenzoic acid (mCPBA) to make biotin derivative 68.

In some embodiments, methods of synthesizing biotin derivatives havingthe structure:

wherein R₁ is a derivative group, is provided. In some embodiments, themethod comprises reacting biotin methyl ester with a secondaryamine-reactive derivative group.

The term “secondary amine-reactive derivative group,” as used hereinrefers to a derivative group comprising a chemical moiety thatfacilitates attachment of the derivative group to a secondary amine. Insome embodiments, the chemical moiety that facilitates attachment of thederivative group to a secondary amine is a leaving group. Nonlimitingexemplary secondary amine-reactive derivative groups include iodoalkyls,bromoalkyls, chloroalkyls, and alkyl chloroformates. One skilled in theart can select a suitable secondary amine-reactive derivative groupaccording to the derivative group to be attached and the particularapplication.

In some embodiments, methods of synthesizing biotin derivatives havingthe structure:

wherein R₂ is a derivative group, are provided. In some embodiments, themethod comprises reacting biotin methyl ester with a secondaryamine-reactive protecting group. In some embodiments, the method furthercomprises reacting the product (i.e., a biotin methyl ester comprising aprotecting group on the nitrogen at the 5 position) with a secondaryamine-reactive derivative group. In some embodiments, the method furthercomprises removing the protecting group.

The term “secondary amine-reactive protecting group,” as used hereinrefers to a protecting group comprising a chemical moiety thatfacilitates attachment of the protecting group to a secondary amine. Insome embodiments, the chemical moiety that facilitates attachment of theprotecting group to a secondary amine is a leaving group. As usedherein, the term “protecting group” refers to a chemical moiety thatprevents a reactive group, such as a secondary amine, from reacting witha reactive species in a mixture, wherein the protecting group can beremoved under conditions that do not otherwise alter the biotinderivative to which it is attached. Nonlimiting exemplary secondaryamine-reactive protecting groups include dimethoxytrityl chloride(DMTr-Cl), trityl chloride (Trt-Cl), benzyl chloroformate (Cbz-Cl), andallyl chloroformate (Aloc-Cl). One skilled in the art can select asuitable secondary amine-reactive protecting group for a particularapplication.

In some embodiments, methods of synthesizing biotin derivatives havingthe structure:

wherein R₁ is a derivative group, are provided. In some embodiments, themethod comprises reacting desthiobiotin methyl ester with a secondaryamine-reactive derivative group.

In some embodiments, methods of synthesizing biotin derivatives havingthe structure:

wherein R₂ is a derivative group, are provided. In some embodiments, themethod comprises reacting desthiobiotin methyl ester with a secondaryamine-reactive protecting group. In some embodiments, the method furthercomprises reacting the product (i.e., a desthiobiotin methyl estercomprising a protecting group on the nitrogen at the 5 position) with asecondary amine-reactive derivative group. In some embodiments, themethod further comprises removing the protecting group.

In some embodiments, a method of synthesizing biotin derivative 79 isprovided.

In some embodiments, the method comprises reacting desthiobiotin methylester with Lawesson's reagent.

In some embodiments, a methyl ester of a biotin derivative is treatedwith a base, such as NaOH, to produce the carboxylic acid of the biotinderivative. In some embodiments, the carboxylic acid of the biotinderivative is modified with a reactive group, such as anN-hydroxysuccinimide (NHS) ester, by reacting the carboxylic acid with acarboxylic acid-modifying reactive group.

A “carboxylic acid-modifying reactive group,” as used herein, refers toa reactive group comprising a chemical moiety that facilitatesattachment of the reactive group to a carboxylic acid. Nonlimitingexemplary carboxylic acid-modifying reactive groups include:

In some embodiments, a suitable linker can be incorporated into a biotinderivative by reacting a biotin derivative comprising an NHS ester witha linker group comprising a primary amine. For example, in someembodiments, a polyethylene glycol linker can be added to a biotinderivative by reacting the NHS ester of the biotin derivative with:

wherein n is an integer from 0 to 20. The carboxylic acid on theresulting biotin derivative may, in some embodiments, be modified with areactive group as described above. Alternatively, a linker thatcomprises a primary amine at one location and a reactive group atanother location may be reacted with a biotin derivative comprising anNHS ester in order to produce a biotin derivative comprising a linkerand a reactive group.

In some embodiments, a reactive group on a biotin derivative is used tolink the biotin derivative to a moiety selected from a protein, apeptide, an amino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle (once linked, the molecule isstill referred to as a “biotin derivative”). Many methods of usingreactive groups to link compounds to such moieties are known in the art.Further, one skilled in the art can select a suitable reactive groupdepending on the moiety that will be linked to the biotin derivative.For example, in some embodiments, an NHS ester reactive group may beused to link a biotin derivative to a moiety comprising a primary amine(such as a peptide, protein, antibody, etc.). In some embodiments, anazide reactive group may be used to link a biotin derivative to a moietycomprising an alkyne by click chemistry. In some embodiments, an alkynereactive group may be used to link a biotin derivative to a moietycomprising an azide by click chemistry.

In some embodiments, a linker of a biotin derivative comprises adetectable moiety. A nonlimiting exemplary method of attaching adetectable moiety to a linker group of a biotin derivative is shown inExample 9.

One skilled in the art can synthesize biotin derivatives of Formulae Ito VI using the methods and reagents discussed herein, and/or themethods and reagents known in the art. It is within the skill in the artto select suitable reagents, reaction conditions, solvents, etc., formaking a particular biotin derivative of any one of Formulae I to VI.

Exemplary Compositions Comprising Biotin Derivatives

In some embodiments, compositions comprising one or more biotinderivatives are provided. In some embodiments, a composition comprisesone or more biotin derivatives in a suitable solvent. Solvents include,but are not limited to, aqueous solvents, nonaqueous solvents, andaqueous/nonaqueous solvent systems. Nonlimiting exemplary solventsinclude water, methanol, ethanol, dimethylsulfoxide (DMSO),dimethylformamide (DMF), dimethylacetamide, and N-methylpyrrolidinone(NMP). In some embodiments, a composition comprises one or more saltsand/or one or more buffering agents. Many salts and buffering agents areknown in the art. One skilled in the art can select a suitable solvent,one or more suitable salts, and/or one or more suitable buffering agentsaccording to the intended application.

In some embodiments, a composition comprising one or more biotinderivatives is dry. That is, in some embodiments, the composition doesnot comprise a solvent, but comprises one or more additional componentsbesides the one or more biotin derivatives. In some embodiments, a drycomposition comprising one or more biotin derivatives comprises one ormore salts and/or one or more buffering agents. In some embodiments, thedry composition is suitable for reconstitution in a solvent, forexample, prior to use.

Exemplary Kits Comprising Biotin Derivatives

In some embodiments, kits comprising one or more biotin derivatives areprovided. Such kits may comprise, in some embodiments, at least onecomposition comprising one or more biotin derivatives. In someembodiments, a composition in a kit is a dry composition, as discussedabove. In some embodiments, a composition in a kit comprises a solvent,as discussed above.

In some embodiments, a kit comprises one or more additional components.Nonlimiting exemplary additional components include sample handlingdevices (such as pipettes, droppers, etc.), containers suitable for usein various instruments (such as cuvettes, microwell plates, etc.),buffers for certain applications (such as reaction buffers, elutionbuffers, binding buffers, etc.), other reagents (such as biotin foreluting the biotin derivatives, streptavidin-coated solid supports, andreagents to facilitate detection), and instructions for use of thebiotin derivatives.

Exemplary Methods of Using Biotin Derivatives

In some embodiments, methods of immobilizing a biotin derivative of anyone of Formulae I to VI are provided. In some embodiments, such methodscomprise contacting the biotin derivative with a solid support thatcomprises a biotin-binding moiety. In some embodiments, methods furthercomprise eluting the biotin derivative of any one of Formulae I to VIfrom the biotin-binding moiety.

A “biotin-binding moiety,” as used herein, refers to a moiety thatspecifically binds biotin. Nonlimiting exemplary biotin-binding moietiesinclude, but are not limited to, avidin, streptavidin, and derivativesof avidin and streptavidin that specifically bind biotin. Nonlimitingexemplary derivatives of avidin and streptavidin include avidins andstreptavidins with one or more amino acid substitutions, deletions,and/or modifications. Nonlimiting exemplary such derivatives aredescribed, e.g., in U.S. Publication No. 2008/0255004; PCT PublicationNo. WO 01/05977; U.S. Pat. Nos. 6,022,951; 6,391,571; 6,312,916;6,417,331; 6,165,750; 6,156,493; 5,973,124; and 5,168,049.

In some embodiments, where later dissociation may be desired, a biotinderivative selected from the biotin derivatives of Formulae I to VI hasan affinity for a biotin-binding moiety that is less than the affinityof biotin for the biotin-binding moiety.

Thus, in some embodiments, a biotin derivative selected from the biotinderivatives of Formulae I to VI binds a biotin-binding moiety with aK_(D) of greater than 1×10⁻¹³ M. In some embodiments, a biotinderivative selected from the biotin derivatives of Formulae I to VIbinds a biotin-binding moiety with a K_(D) of greater than 1×10⁻¹³ M. Insome embodiments, a biotin derivative selected from the biotinderivatives of Formulae I to VI binds a biotin-binding moiety with aK_(D) of between 1×10⁻¹³ M and 1×10⁻⁸ M, between 1×10⁻¹² M and 1×10⁻⁹ M,or between 1×10⁻¹¹ M and 1×10⁻⁹ M, between 10⁻¹³ M to 10⁻⁴ M, between10⁻¹² M to 10⁻⁴M, between 10⁻¹¹ M to 10⁻¹¹ M, between 10⁻¹⁰ M to 10⁻⁴ M,between 10⁻⁹ M to 10⁻⁴ M, between 10⁻⁸ M to 10⁻⁴ M, between 10⁻⁷ M to10⁻⁴ M, or between 10⁻⁶ M to 10⁻⁴ M.

In some embodiments, where later dissociation is not desired, a biotinderivative selected from the biotin derivatives of Formulae I to VI hasan affinity for a biotin-binding moiety that is less than or equal to1×10⁻¹³ M. Thus, in some embodiments, a biotin derivative selected fromthe biotin derivatives of Formulae I to VI binds a biotin-binding moietywith a K_(D) of between about 1×10⁻¹³ M to about 1×10⁻¹⁵ M.

Nonlimiting exemplary solid supports include polymers (such as agarose,sepharose, cellulose, nitrocellulose, alginate, Teflon, latex,acrylamide, nylon, plastic, polystyrene, silicone, etc.), glass, silica,ceramics, and metals. Such solid supports may take any form, such asparticles (including microparticles), sheets, dip-sticks, gels, filters,membranes, microfiber strips, tubes, wells, plates (such as microplates,including 6-well plates, 24-well plates, 96-well plates, 384-wellplates, etc.), fibers, capillaries, combs, pipette tips, microarraychips, etc. In some embodiments, the biotin-binding moiety is associatedwith the surface of a solid support. In some embodiments, the surface ofthe solid support comprises an irregular surface, such as a porous,particulate, fibrous, webbed, or sintered surface.

In some embodiments, a solid support is selected from a microplate, amicroarray chip, and a microparticle. In some embodiments, a solidsupport is at least partially composed of a polymer. In someembodiments, a microparticle solid support comprises monodisperse orpolydisperse spherical beads. Monodisperse microparticles aresubstantially uniform in size (i.e., they have a diameter standarddeviation of less than 5%), while polydisperse microparticles vary insize. In some embodiments, microparticles are composed of the samepolymer throughout, or are core-shell polymers, in which the core of themicroparticle is composed of one polymer, and the outer layer (or“shell”) is composed of another. In some embodiments, microparticles aremagnetic.

In some embodiments, a biotin-binding moiety is attached to a solidsupport through an amino or sulfhydryl group of the biotin-bindingmoiety. In some such embodiments, the surface of the solid supportcomprises a group capable of reacting with a free amine or sulfhydrylgroup. Nonlimiting exemplary such groups include carboxy, activehalogen, activated 2-substituted ethylsulfonyl, activated 2-substitutedethyl carbonyl, active ester, vinylsulfonyl, vinylcarbonyl, aldehyde,epoxy, etc. Some such groups may require the use of an additionalreactant to render the group capable of reacting with a free amine orsulfhydryl group. Nonlimiting exemplary additional reactants includecyanogen bromide, carbonyldiimidazole, glutaraldehyde,hydroxylsuccinimide, tosyl chloride, etc.

Many solid supports are known in the art, and one skilled in the art canselect a suitable solid support according to the intended application.Similarly, if the solid support is not commercially available with abiotin-binding moiety attached to its surface, one skilled in the artcan select a suitable method of attaching a biotin-binding moiety to asolid surface. Exemplary such methods are described, e.g., in U.S.Publication No. US 2008/022004 A1.

In some embodiments, eluting comprises contacting the immobilized biotinderivative with a displacement molecule. A “displacement molecule” asused herein is a molecule that has an affinity for the biotin-bindingmoiety that is greater than the affinity of the biotin derivative forthe biotin-binding moiety. A nonlimiting example of such a molecule isbiotin. The term “biotin” as used herein includes native biotin andnon-native biotins, such as biotin methyl ester and biotin multimers,including, but not limited to, biotin dimers and biotin trimers.Nonlimiting exemplary biotins include the nonnative biotins described,e.g., in Wilbur et al., Bioconjugate Chem. 8: 819-32 (1997). In someembodiments, a biotin binds to the biotin-binding moiety with a K_(D) ofless than 1×10⁻¹⁴ M. In some embodiments, a nonnative biotin is moresoluble in aqueous solvents than native biotin. The term “native biotin”as used herein refers to a biotin having the structure:

In some embodiments, eluting comprises exposing the immobilized biotinderivative to concentrations of native biotin or non-native biotins,such as biotin multimers, sufficient to cause release, such as, forexample, millimolar (mM) concentration, at room temperature or at 37° C.with mixing.

In some embodiments, the methods described herein are used for thedetection, identification, determination, purification, separation,and/or isolation of an entity from a sample. In some embodiments, theentity is attached to a biotin derivative such that it becomes the R₃group in any one of Formulae I to VI. In some embodiments, the entity isselected from a protein, a peptide, an amino acid, a dextran, amonosaccharide, a disaccharide, a trisaccharide, an oligosaccharide, apolysaccharide, a hormone, a lipopolysaccharide, a nucleotide, anoligonucleotide, a small molecule, a cell, or a microparticle. In someembodiments, R₃ is a moiety that binds to the entity to be separatedfrom a sample.

In some embodiments, R₃ is an activity-based probe, which specificallyinteracts with a particular entity or class of entities, and, in someembodiments, becomes covalently attached to an entity upon binding.Nonlimiting exemplary activity-based probes are described, e.g., inBachovchin et al., Nat. Biotech. 27: 387-394 (2009); Cravatt et al.,Ann. Rev. Biochem. 77: 383-414 (2008); Fonovid et al., Curr. Pharmac.Des. 13: 253-261 (2007); Kato et al., Nat. Chem. Biol. 1: 33-38 (2005);Patricelli et al., Biochem. 46: 350-358 (2007); Paulick et al., Curr.Opin. Genet. Dev. 18: 97-106 (2008); Saghatelian et al., PNAS 101:10000-10005 (2004); Salisbury et al., J. Am. Chem. Soc. 130: 2184-2194(2008); Salisbury et al. PNAS 104: 1171-1176 (2007); Wright et al.,Chem. & Biol. 14: 1043-1051 (2007); Wright et al., JACS 131: 10692-10700(2009); U.S. Pat. No. 6,872,574 B2; and U.S. Publication Nos. US2009/0252677 A1 and US 2008/0176841 A1.

In some such embodiments, R₃ is an antibody that binds an entity in asample. Thus, in some embodiments, a biotin derivative of any one ofFormulae I to VI, wherein R₃ is, for example, an antibody or otherentity-binding moiety, is used in the described methods to bind to theentity in a sample. The entity may then be detected, identified,determined, purified, separated, and/or isolated as desired.

In some embodiments, the sample is a biological sample. Such biologicalsamples include, but are not limited to, blood and blood fractions andproducts, saliva, lymph, bile, urine, milk, feces, spinal fluid, semen,cells, proteins, cells comprising target proteins therein or thereon,cell extracts, fluid preparations of tissue samples, organ biopsysamples, etc. Other exemplary samples include, but are not limited to,water samples, fluid preparations of soil samples, food samples, etc.

In some embodiments, the methods described herein are used to detect,identify, determine, purify, separate, and/or isolate cells and/orviruses from a sample. Nonlimiting exemplary cells include prokaryoticand eukaryotic cells, including mammalian cells, non-mammalian animalcells, plant cells, insect cells, fungal cells, yeast cells, protozoa,bacteria, etc. Nonlimiting exemplary viruses include DNA and RNA virusesand retroviruses, etc. In some embodiments, the cells and/or virusesremain viable throughout the method. In some embodiments, the cells orviruses are propagated following separation, purification, and/orisolation using the method. In some embodiments, the cells and/orviruses are considered to have “remained viable” if, followingapplication of the method, at least 75%, at least 80%, at least 85%, orat least 90% of the cells and/or viruses are viable (i.e., capable ofpropagating and/or carrying out cellular or viral processes).

In some embodiments, binding of the biotin derivative of any one ofFormulae I to VI to an entity is used to label the entity, e.g., fordetection. In some embodiments, the biotin derivative of any one ofFormulae I to VI binds to the entity through the R₃ group, whichcomprises an entity-binding moiety. In some embodiments, the biotinderivative of any one of Formulae I to VI comprises a detectable moietyas part of a linker (or L group). Thus, in some such embodiments,following binding, the entity can be detected by association of thedetectable moiety. Such detection may occur with or without (or beforeor after) purification, separation, and/or isolation of the entity bythe methods described herein (such as binding the biotin derivative to abiotin-binding moiety).

As a non-limiting example, a biotin derivative of any one of Formulae Ito VI comprising a linker with a detectable moiety and an R₃ group thatbinds to a factor on the surface of a cell may be bound to the cell in amixture. Following binding, the mixture may be examined under amicroscope to detect association of the detectable moiety with cells inthe mixture. The cells may then be contacted with a microparticlecomprising a biotin-binding moiety on the surface. The biotin-bindingmoiety may bind to the biotin derivative of any one of Formulae I to VI,resulting in association of the microparticle with the cells in themixture. The cells may then be separated from the mixture by someproperty of the microparticles (such as, for example, by magneticseparation or gentle centrifugation). The biotinderivative/biotin-binding moiety interaction may then be interrupted(i.e., using an elution step). The cell will remain associated with thedetectable moiety, but not with the microparticle. The cell can then bemanipulated as desired, in some embodiments, with the detectable moietystill associated.

The claimed invention is in no way limited to the embodimentsexemplified herein. One skilled in the art can envision many additionalapplications of the described biotin derivatives and methods.

Examples

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1: Preparation of 2,5-dioxopyrrolidin-1-yl6-(5-((3aS,4S,6aR)-5,5-dioxido-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoate(biotin derivative 1)

Biotin methyl ester 62 was oxidized to sulfone 63 using hydrogenperoxide, and then the methyl ester was hydrolyzed to free acid 64,which was converted to NHS ester 65. The NHS ester 65 was reacted with6-aminocaproic acid to give 66, which was converted to NHS ester 1.

Example 2: Preparation of 2,5-dioxopyrrolidin-1-yl5-((3aS,4S,6aR)-5,5-dioxido-2-thioxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate(biotin derivative 2)

Biotin methyl ester 62 was converted to 67 using Lawesson's reagent(2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide).The methyl ester 67 was oxidized to sulfone 68 using hydrogen peroxide.The methyl ester 68 was hydrolyzed to free acid 69, which was thenconverted to NHS ester 2.

Example 3: Preparation of 2,5-dioxopyrrolidin-1-yl5-((3aS,4S,6aR)-2-thioxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate(biotin derivative 3)

Biotin methyl ester 62 was converted to 67 using Lawesson's reagent. Themethyl ester 67 was hydrolyzed to free acid 70, which was then convertedto NHS ester 3.

Example 4: Preparation of 2,5-dioxopyrrolidin-1-yl5-((3aS,4S,6aR)-1-methyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate(biotin derivative 4)

Biotin methyl ester 62 was selectively alkylated by iodomethane to give71. The methyl ester 71 was hydrolyzed to free acid 70, which was thenconverted to NHS ester 4.

Example 5: Preparation of 2,5-dioxopyrrolidin-1-yl5-((3aS,4S,6aR)-3-ethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate(biotin derivative 6)

Biotin methyl ester 62 was selectively protected by dimethoxytritylchloride (DMTr-Cl) to give 73, followed by alkylation with iodoethane toyield 74. The DMTr protecting group was cleaved by 80% acetic acid togive 75. The methyl ester 75 was hydrolyzed to free acid 76, which wasthen converted to NHS ester 6.

Example 6: Preparation of 2,5-dioxopyrrolidin-1-yl21-((3aS,4S,6aR)-3-ethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-17-oxo-4,7,10,13-tetraoxa-16-azahenicosan-1-oate(biotin derivative 17)

Biotin derivative 6 as prepared above was reacted with amino-(PEG)₄-acidto yield 77, which was then converted to NHS ester 17.

Example 7: Preparation of 2,5-dioxopyrrolidin-1-yl6-((4R,5S)-5-methyl-2-thioxoimidazolidin-4-yl)hexanoate (biotinderivative 18)

DSB methyl ester 78 was converted to 79 by Lawesson's reagent. Themethyl ester 79 was hydrolyzed to free acid 80, which was then convertedto NHS ester 18.

Example 8: Preparation of 2,5-dioxopyrrolidin-1-yl22-((4R,5S)-5-methyl-2-thioxoimidazolidin-4-yl)-17-oxo-4,7,10,13-tetraoxa-16-azadocosan-1-oate(biotin derivative 20)

DSB NHS ester 81 was reacted with amino-(PEG)₄-acid tert-butyl ester toyield 82, which was converted to 83 by Lawesson's reagent. Thetert-butyl ester was deprotected by TFA to give free acid 84, which wasthen converted to NHS ester 20.

Example 9: Preparation of2-(6-amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-(((S)-23-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-1-((3aS,4S,6aR)-3-ethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-5,21-dioxo-9,12,15,18-tetraoxa-6,22-diazaheptacosan-27-yl)carbamoyl)benzoate(biotin derivative 21)

Biotin derivative 17 as prepared above was reacted withL-Lys(Boc)-O^(t)Bu, followed by deprotection by TFA to give biotinderivative 85. Then biotin derivative 85 was reacted with AF488, 5-NHSester, followed by conversion to NHS ester 21.

Example 10: Preparation ofN-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-3-ethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide(biotin derivative 23)

Biotin derivative 6 as prepared above was reacted with compound 86 togive biotin derivative 23.

Example 11: Preparation of5-((3aS,4S,6aR)-3-ethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-N-(3,6,9,12-tetraoxapentadec-14-ynyl)pentanamide(biotin derivative 24)

Biotin derivative 6 as prepared above was reacted with compound 87 togive biotin derivative 24.

Example 12: Preparation of Antibody-Linked Biotin Derivatives

A series of biotin derivative conjugates of goat anti-mouse IgG (GAM)were prepared by standard methods. See, e.g., Haugland et al., Meth.Mol. Biol. 45: 205 (1995). The goat anti-mouse antibody was labeled withAlexa Fluor® 488 Dye (Invitrogen) prior to conjugation to the biotinderivatives to facilitate later detection. An exemplary method forprotein conjugation with succinimidyl esters of the invention is asfollows. One skilled in the art can vary the ratios of biotin derivativeto protein, protein concentration, time, temperature, buffercomposition, etc. while making a desired conjugate. A solution ofprotein is prepared at ˜10 mg/mL in 0.1 M sodium bicarbonate. The biotinderivative is dissolved in a suitable solvent such as DMF or DMSO at ˜10mg/mL. Predetermined amounts of the biotin derivative is added to theprotein solution with stirring. A molar ratio of 20 equivalents ofbiotin derivative to 1 equivalent of protein is typical, though theoptimal amount varies with the particular biotin derivative, and can bedetermined by one skilled in the art. The reaction mixture is incubatedat room temperature for one hour or on ice for several hours. The biotinderivative-protein conjugate is typically separated from free unreactedreagents by size-exclusion chromatography, such as on a Bio-Rad P-30resin equilibrated with phosphate-buffer saline (PBS).

Antibody-linked biotin derivatives are referred to as “1-Ab,” “2-Ab,”“3-Ab,” etc. The structures of the antibody-linked biotin derivativesare shown below in Table 5.

Example 13: Analysis of Capture and Detachment Efficiency ofAntibody-Linked Biotin Derivatives

One-half ml of antibody-linked biotin derivative (0.1 mg/ml in PBSbuffer) was incubated with 100 μl of M280 Streptavidin Dynabeads (10mg/ml) and mixed at RT for 10 minutes. The tube was placed on a magnetfor 1 minute. The supernatant was carefully removed. The tube was thenremoved from the magnet, and 0.5 ml PBS buffer was added, and the beadswere resuspended by gentle pipetting 5 times. The tube was placed on themagnet for 1 minute. The supernatant was carefully removed. The PBSbuffer wash was repeated twice. The tube was removed from the magnet andthe beads were carefully resuspended in 0.5 ml of fresh elution buffercontaining 5 mM biotin or 5 mM bis-biotin in PBS, and mixed at roomtemperature for 5 minutes. The tube was placed on the magnet for 1minute. The supernatant was carefully removed. The efficiencies ofcapture and detachment were measured by the fluorescence intensity ofthe supernatant.

The results of that experiment are shown in Table 5.

TABLE 5 Capture and detachment efficiency of antibody-lined biotinderivatives De- Cap- tach- Biotin derivative ture ment

DSB- X-Ab  97% 16%

1- Ab  60% 35%

2- Ab  17%  8%

3- Ab  86% 75%

4- Ab  55% 85%

114- Ab  84% 76% 2,5-dioxopyrrolidin-1-yl 3-(5-((3aS,4S,6aR)-  0% n/a1,3-dimethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)propanoate-Ab

5- Ab  82% 64%

6- Ab  94% 84%

9- Ab  70% 85%

10- Ab  76% 92%

11- Ab  90% 92%

12- Ab  98% 95%

113- Ab  94% 65%

14- Ab  98% 60%

15- Ab 100% 55%

17- Ab  97% 100% 

18- Ab  75% 85%

19- Ab  84% 92%

20- Ab  92% 95%

21- Ab  97% 99%

22- Ab  94% 95%

Example 14: Effect of Molar Ratio of Labeling on Capture and DetachmentEfficiency

Antibodies were labeled with biotin derivatives 14, 17, and 20, usingthe method described in Example 12. The labeling was carried out atvarious initial molar ratios of the biotin derivative to antibody. Themethod described in Example 13 was then used to determine the effect ofinitial molar ratio of labeling on capture and detachment efficiency.

The results of that experiment are shown in Table 6.

TABLE 6 Capture and detachment efficiency of varying labeling molarratio of biotin derivatives to antibody Initial Molar Ratio (biotinderivative to Cap- De- Biotin derivative antibody) ture tach.

14-Ab  5 97% 78% 14-Ab 10 98% 75% 14-Ab 20 97% 62%

20-Ab  5 55% 70% 20-Ab 10 70% 75% 20-Ab 20 80% 76% 20-Ab 30 94% 96%

17-Ab  5 75% 96% 17-Ab 10 80% 94% 17-Ab 20 97% 99% 17-Ab 30 100%  97%

The results show that initial molar ratios of labeling, from about 10 toabout 30, has little effect on the capture and detachment efficiency forbiotin derivatives 14 and 17. For biotin derivative 20, increasing themolar ratio of labeling can increase the capture and release efficiency.

Example 15: Determination of Rate Constants and Binding Affinity UsingBioLayer Interferometry

In BioLayer Interferometry (BLI), a layer of molecules bound to the tipof an optic fiber creates an interference pattern in white lightreflected from the layer of molecules. A change in the number ofmolecules bound to the tip of the optic fiber causes a shift in thatinterference pattern that can be measured. The wavelength shift is adirect measure of the thickness of the layer of molecules. Further,because the shift can be measured in real-time, rates of association anddissociation can be determined. See, e.g., Concepcion et al.,Combinatorial Chemistry and High Throughput Screening, 12, 791-800(2009); Lotze, et al., United States Patent Application 20090147264;Abdiche et al., Anal. Biochem., 377(2), 209-217 (2008).

The rate constants and binding affinity of certain biotin derivativesfor streptavidin were determined using BLI, as follows. The streptavidinbiosensor tips from ForteBio were prewet for 30 min in water prior touse. All interaction analyses were conducted at 25° C. in kineticbuffer. The 96-well microplates used in the Octet were filled with 200μl of samples or buffer per well and agitated at 1000 rpm. Threedifferent concentrations of antibody-linked biotin derivative sampleswere added to 96-well microplate in duplicate, and a buffer solution wasalso added to 96-well microplate as a reference control. The associationcurve was generated by dipping the streptavidin biosensor tips into thesample well and incubated at 25° C. for 500 seconds at 1000 rpm. Thedissociation curve was generated by dipping the streptavidin biosensortips into the kinetic buffer well and incubated at 25° C. for 500seconds at 1000 rpm. The kinetic data was obtained using the DataAnalysis Octet software.

The results of that experiment are shown in Table 7

TABLE 7 Binding constants of antibody-linked biotin derivatives BiotinK_(on) K_(d) derivative Capture Detach. K_(D) (M) (1/Ms) (1/sec)Biotin-Ab 100% 0% 5.95 × 10⁻¹⁵ 2.41 × 10⁷ 1.43 × 10⁻⁷ DSB-Ab 98% 10%3.54 × 10⁻¹³ 1.30 × 10⁶ 4.58 × 10⁻⁷ 1-Ab 60% 35% 1.26 × 10⁻¹⁰ 1.30 × 10⁵1.65 × 10⁻⁵ 3-Ab 86% 75% 4.54 × 10⁻¹⁰ 1.56 × 10⁵ 7.10 × 10⁻⁵ 4-Ab 55%85% 3.92 × 10⁻⁹  1.03 × 10⁴ 4.04 × 10⁻⁵ 10-Ab 76% 92% 1.52 × 10⁻⁹  1.73× 10⁵ 2.63 × 10⁻⁴ 14-Ab 98% 60% 1.44 × 10⁻¹¹ 3.43 × 10⁵ 4.91 × 10⁻⁶21-Ab 97% 99% 2.56 × 10⁻¹¹ 3.43 × 10⁵ 4.91 × 10⁻⁶ 22-Ab 94% 95% 3.34 ×10⁻⁹  7.85 × 10⁴ 2.62 × 10⁻⁴ 11-Ab 90% 92% 5.65 × 10⁻¹⁰ 1.14 × 10⁵ 6.42× 10⁻⁵ 12-Ab 98% 95% 8.79 × 10⁻¹² 3.61 × 10⁵ 3.17 × 10⁻⁶

The results show that a dynamic diversity of biotin derivatives has beendeveloped with the binding affinity range from 1×10⁻¹³ M to 1×10⁻⁸ M.Desired capture and detachment efficiencies can therefore be obtained bychoosing appropriate biotin derivatives.

Example 16: Isolation of Antibody-Linked Biotin Derivative from CellLysate

To determine if a tunable biotin derivative will function in a celllysate, goat anti-mouse IgG/biotin derivative 21 conjugate was chosen tovalidate the protein purification from cell lysate. The cell lysate wasprepared by suspending the Jurkat cells (˜3×10⁷) in 1 ml of Tris Buffercontaining 0.5% SDS and sonicating for 1 minute to make about 2 mg/mltotal protein. The goat anti-mouse IgG conjugate 21-Ab (100 μg) wasmixed with 0.5 ml of cell lysate and vortexed for 30 seconds. 100 μl ofstreptavidin coated agarose was added, and incubated for 5 minutes atroom temperature under rolling and tilting, followed by centrifugationfor 3 minutes at 10,000×g. The supernatant was removed. One ml PBSbuffer was added to the streptavidin beads, and mixed for 3 minutes atRT, followed by centrifugation for 3 minutes at 10,000×g. Thesupernatant was removed. The PBS washing step was repeated twice. 500 μlof 3 mM biotin solution was added to the streptavidin beads andincubated for 5 minutes at RT under rolling and tilting, followed bycentrifugation for 3 minutes at 10,000×g. The goat anti-mouse IgGconjugate 21-Ab was recovered in the supernatant. The recoveryefficiency was measured by the fluorescent intensity. The purity ofisolated protein was checked by protein gel stain.

The results are shown in FIG. 1A and FIG. 1B. FIG. 1A shows thefluorescence labeled protein distribution in the tube before the captureand after the capture, then after release from streptavidin coatedagarose. FIG. 1B shows the protein gel stain of cell lysate mixture,supernatant, washing, and purified protein.

Example 17: Isolation of T Cells from Peripheral Blood Mononuclear Cells

To determine if an antibody-linked biotin derivative can be used toisolate CD3+ T cells, an anti-CD3 antibody was labeled with biotinderivative 17. Peripheral blood mononucleocytes (PBMCs) were isolatedfrom anti-coagulated peripheral blood or leukocyte enriched buffy coatusing standard procedure. Isolation Buffer (Ca²⁺ and Mg²⁺ free phosphatebuffered saline (PBS) supplemented with 0.1% BSA and 2 mM EDTA) wasprepared. The PBMCs were suspended with Isolation Buffer at 1×10⁸cells/ml. 500 μl (˜5×10⁷ cells) of the PBMCs suspension, was mixed with25 μl anti-CD3 antibody labeled with biotin derivative 17 for 10 minutesat 2-8° C. Two ml Isolation Buffer was added to wash cell, followed bycentrifugation for 8 minutes at 350×g. The supernatant was removed anddiscarded. One ml Isolation Buffer was added and the cell pellet wasresupended.

75 μl of resuspended M280 Streptavidin Dynabeads (10 mg/ml) was addedand mixed at room temperature for 15 minutes. The tube was placed on themagnet for 1 minute. The supernatant was carefully removed anddiscarded. The tube was removed from the magnet, and 1 ml IsolationBuffer was added, and the bead-bound cells were suspended by gentlepipetting 5 times. The tube was placed on the magnet for 1 minute. Thesupernatant was carefully removed and discarded. The tube was removedfrom the magnet and the bead-bound cells was resuspended in 1 ml offresh 5 mM biotin buffer in Isolation Buffer, and incubated for 10minutes at room temperature under rolling and tilting. The tube wasplaced on the magnet for 1 minute, and the supernatant containing thebead-free cells was transferred to a new tube, and was again placed onthe magnet for 1 minute to remove any residual beads. The supernatantcontaining the bead-free cells was transferred to a new tube, and 2 mlIsolation Buffer was added, followed by centrifugation for 8 minutes at350×g. The supernatant was discarded and the cell pellet was resuspendedin preferred medium (such as MEM, DMEM, or RPMI). The viability andpurity of isolated CD3+ T cells was checked by Flow Cytometry.

The results are shown in FIG. 2. FIG. 2, Step A shows the mononuclearcell pool incubated with anti-CD3 antibody labeled with biotinderivative 17, which is then bound to M280 Streptavidin Dynabeads. FIG.2, Step B shows the isolated CD3+ T cells following dissociation ofbiotin derivative 17 from the M280 Streptavidin Dynabeads. FIG. 2, StepC shows the free M280 Streptavidin Dynabeads after elution with biotinsolution. In that experiment, 100% of the CD3+ T cells were depletedfrom the mononuclear cell pool, as determined by flow cytometry.Further, 80% of the CD3+ T cells were recovered, and the recovered CD3+T cells were 99% pure.

Example 18: Preparation of AlexaFluor 488 Derivative of Compound 17

Example 19: Binding Affinity and Reversibility of Binding of Compound117 with Strepavidin

In order to evaluate the binding affinity and reversibility of N3′-ethylbiotin with streptavidin, a simple fluorescent binding and release assaywas designed. Briefly, N3′-ethyl biotin-AF488 conjugate 117 wasincubated with M-280 Streptavidin Dynabeads in pH 7.4 PBS buffer. After25 min incubation at room temperature, the magnetic beads were separatedfrom the supernatant, which was used to measure the capture efficiency,by measuring the fluorescent intensity of the supernatant solution todetermine the amount that was not captured. Then, the N3′-ethyl biotin117 bound magnetic beads were washed with PBS buffer to remove weak ornon-specific binding. The reversibility was tested by incubating theN3′-ethyl biotin 117 bound magnetic beads with 2 mM biotin in PBS bufferat room temperature for 5 min, then the magnetic beads were concentratedby a magnet, and the fluorescent intensity in the solution was measuredto determine the amount that was released. As a control, biocytin-AF488conjugate was also tested in the same assay format. As shown below, theN3′-ethyl biotin displays high binding efficiency and the binding isfully reversible with biotin competing reagent under non-denaturingconditions.

Compound Capture efficiency Release efficiency Biocytin 96 ± 2% <5% 11794 ± 3% 95 ± 4%

The details of fluorescent capture and release assay were follows. 100μL of M280 Streptavidin Dynabeads (10 mg/mL, Invitrogen) was transferredto a 1.5 mL centrifuge tube, and washed with 500 μL PBS three times. 500μL of N3′-Ethyl biotin-AF488 conjugate 117 (0.2 μM in PBS buffer, pH7.4) was added and mixed under rolling and tilting at room temperaturefor 25 minutes. The tube was placed on the magnet for 1 minute, toconcentrate the magnetic beads on the magnet. The supernatant wascarefully removed and saved for measuring the capture efficiency, bymeasuring the fluorescent intensity of the supernatant solution. Thetube was removed from the magnet, and 500 μL PBS was added, then thebeads were resuspended by gentle pipetting 5 times. The tube was placedon the magnet for 1 minute. The supernatant was carefully removed. ThePBS washing was repeated one more time. The tube was removed from themagnet and the beads were carefully resuspended in 500 μL of 2 mM biotinin PBS (pH 7.4), with mixing under rolling and tilting at roomtemperature for 5 minutes. The tube was placed on the magnet for 1minute. The supernatant was carefully removed and saved for measuringthe release efficiency, by measuring the fluorescent intensity of thesupernatant solution.

Example 20: Kinetic Measurement of Rate Constants

The binding constant and on-and-off rate constant of N3′-ethyl biotinwith streptavidin were tested using a label-free ForteBio system. TheN3′-ethyl biotin 117 was covalently immobilized to amine reactive sensortips from ForteBio. To generate the association curve, the N3′-ethylbiotin labeled biosensor tips were incubated with streptavidin at 25° C.The dissociation curve was determined by incubating the streptavidinbound sensor tips in PBS buffer described above, as well as in PBSbuffer containing 2 mM biotin at 25° C. As shown in FIG. 3, theN3′-ethyl biotin shows fast on-rate with streptavidin (k_(on)˜1.5×10⁵M⁻¹S⁻¹), and relative slow off-rate from streptavidin in PBS buffer(k_(off)˜1.2×10⁴ S⁻¹). The dissociation constant k_(D) of N3′-ethylbiotin is about 0.8 nM. In the presence of 2 mM biotin, the N3′-ethylbiotin displays fast off-rate (k_(off)˜5.8×10⁻² S⁻¹), thus leadscomplete dissociation from streptavidin.

The details of fluorescent capture and release assay were follows. Allinteraction analyses were conducted at 25° C. in kinetic buffer (PBS,0.1% BSA, 0.05% Tween-20, pH 7.2). The amine reactive biosensor tipsfrom ForteBio were prewet for 20 min in water prior to use. The preparedsamples and buffers were added to a 96-well microplate at 200 μL perwell according the following sample layout, and the entire 96-well platewas agitated at 1000 rpm. Eight biosensor tips were performed thekinetic assay in parallel. Each amine reactive biosensor tip wasequilibrated in MES buffer for 5 min, then activated with EDC/NHS for 5min. The activated biosensor tip was labeled with 2 mM N3′-ethyl biotin117 for 10 min, then quenched with 1 M ethanolamine for 5 min. TheN3′-ethyl biotin 117 immobilized biosensor tip was incubated in kineticbuffer for 5 min to generate baseline, then incubated with 2 μg/mLstreptavidin in kinetic buffer or kinetic buffer (control) at 1000 rpmfor 10 min to generate association curve, followed by incubation withkinetic buffer or 2 mM biotin in kinetic buffer at 1000 rpm for 10 minto generate dissociation curve. The ForteBio Data Analysis Octetsoftware was used to process the curve by subtracting sample curve fromreference curve, and the kinetic data was calculated by fitting curve.

Example 21: Preparation of Propargyl Derivative of Compound 21

Example 22: Use of N3′-Ethyl Biotin Alkyne 118 for Labeling, Detectionand Isolation in a Biological System

The azido sugar labeling system was used to demonstrate the ability ofN3′-ethyl biotin alkyne 118 for efficient labeling, detection, andisolation in a biological system.

Protein Labeling and Detection in Cells.

HeLa cells were cultured on glass coverslips in 10 mL Minimum EssentialMedium (MEM) supplemented with 10% fetal bovine serum (FBS) at four 100mm tissue culture dishes. To each tissue culture dish, 10 μL ofAc₄GlcNAz (100 mM in DMSO), Ac₄GalNAz (100 mM in DMSO), Ac₄ManNAz (100mM in DMSO; Life Technologies, San Diego, Calif., SKU #s C33367, C33367and CC33366, respectively), and DMSO were added, respectively. Thedishes were incubated at 37° C. humidified incubator with 5% CO₂ for 24h. After incubation, the media was removed, and the HeLa cells werewashed with 10 mL DPBS with 1% FBS twice. Then, the HeLa cells werefixed with 3.7% formaldehyde in PBS for 15 min at room temperature andwashed with 10 mL DPBS with 1% FBS once. The HeLa cells werepermeabilized with 0.5% Triton X-100 in PBS for 15 min at roomtemperature, then washed with 10 mL DPBS with 1% FBS once. The HeLacells were labeled with 10 μM of N3′-ethyl biotin alkyne 118 in thepresence of 100 M CuSO₄, 500 μMtris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and 2.5 mM sodiumascorbate in DPBS with 1% FBS at room temperature for 30 min. Forcontrol experiment, the HeLa cells were also labeled with 10 μM ofN3′-ethyl biotin alkyne 118 without CuSO₄. After labeling, the HeLacells were washed again with DPBS with 1% FBS twice, and incubated witha nuclear stain Hoechst 33342 (1:5000 dilution) in DPBS with 1% FBS for10 min. The cells were washed with DPBS with 1% FBS three times, andeH₂O once.

After the coverslips were air-dried, the coverslips were mounted inCytoseal onto microscopy slides. Fluorescent images were captured on aZeiss Axioskop 2 fluorescence microscope with 40× objective equippedwith a Hamamatsu ORCA-ER CCD camera using excitation and emissionfilters from Omega Optical. The nuclear stain Hoechst 33342 was imagedusing a 365±5 nm band-pass filter for excitation and a 400±5 nm cutofffilter for emission. The Alexa Fluor 488 fluorophore was imaged using a480±10 nm band-pass filter for excitation and a 510±10 nm band-passfilter for emission. Exposure times of 20 and 50 ms, for the nuclearstain and Alexa Fluor 488 (AF488) dye channels, respectively, were usedfor image collection. All raw images were processed using Slidebooksoftware with identical leveling.

The cells incubated with azido sugars show increased fluorescence signalwhen compared to control cells that were not incubated with azido sugar,as shown in FIG. 4 and, indicating the labeling of cells with N3′-ethylbiotin alkyne 118 via formation of triazole linkage. The enhancement offluorescence signal is only observed in the presence of both catalystCu(I) and azido glycans. The Cu(I) chelating ligand, THPTA, facilitatesthe reaction and protects protein damage, but is not necessary forlabeling of cells. Cells incubated with Ac4MaNAz supplemented media(top) or unsupplemented media (bottom), as shown in FIG. 4, were labeledN3′-ethyl biotin alkyne 118 in the presence of Cu(I)/THPTA as catalyst.Fluorescence signal from AF488 (left), the nuclear stain, Hoechst 33342,(center) and the merged images (right) are shown. These resultsdemonstrate N3′-ethyl biotin alkyne 118 is an efficient reagent forlabeling and detection in cells.

Protein Isolation.

In order to isolate azido sugar containing glycoproteins from cellmixture, the Jurkat cells were cultured in Ac₄GlcNAz supplemented mediafor 24 h. In this culturing, Jurkat cells were cultured in 100 mL RPMI1640 media supplemented with 10% FBS at two T175 tissue culture flasks.When cell density reached ˜10×10⁶ cells/mL, 100 μL of Ac₄GlcNAz (100 mMin DMSO) and DMSO (control) were added, respectively. The flasks wereincubated at 37° C. humidified incubator with 5% CO₂ for 24 h. Afterincubation, the Jurkat cells were collected by centrifugation at 600×gfor 5 min, and washed with PBS five times. The cell pellet was suspendedin PBS to adjust the cell density at 50×10⁶ cells/mL, and transferred 1mL each to 1.5 mL tube. The cells were collected again by centrifugationat 600×g for 5 min. Then, 1 mL of 0.5% SDS/PBS was added to each tube,and lysated by sonication. The cell lysate was prepared at ˜3 mg/mL in0.5% SDS/PBS.

Cell lysate was incubated with N3′-ethyl biotin alkyne 118 or biotinalkyne in the presence of CuSO₄, THPTA, and sodium ascorbate at roomtemperature as described below. N3′-Ethyl biotin alkyne 118 and biotinalkyne (Invitrogen) were used to perform parallel protein enrichmentexperiments. Biotin alkyne has the structure:

The cell lysate (500 μL, Ac₄GlcNAz treated or untreated) were incubatedwith 50 M of N3′-ethyl biotin alkyne 118 or biotin alkyne in thepresence of 0.5 mM CuSO₄, 2.5 mM THPTA, and 5 mM sodium ascorbate in0.5% SDS/PBS at room temperature for 1 h, respectively. For controlexperiment, the cell lysate were also incubated with 50 μM of N3′-ethylbiotin alkyne 118 or biotin alkyne without CuSO₄, respectively.

After click labeling, the proteins were enriched with streptavidinagarose at room temperature. After incubation to perform the clicklabeling, 500 μL MeOH and 150 μL CHCl₃ were added to each tube. The tubewas vortexed and centrifuged at 10,000 rpm for 4 min. The solution wasremoved and the pellet was washed with MeOH five times, thenresolubilized in 500 μL of 0.5% SDS/PBS. 200 μL of Streptavidin agarose(Invitrogen) was added to each tube and incubated at room temperaturefor 1 h. The agarose beads were transferred to 2 mL spin column(Bio-Rad), and washed with 2 mL TEST (10 mM Tris, 1 mM EDTA, 1 M NaCl,0.1% Tween-20, pH 7.4) five times. After washing step, the proteinslabeled with N3′-ethyl biotin alkyne 118 were eluted out fromstreptavidin agarose using 2 mM biotin or 10 mM HCl in H₂O (1 mL×3); theproteins labeled with biotin alkyne were eluted out by heatingprotein-bound streptavidin agarose in 1% SDS in H₂O at 95° C. for 5 min.The eluate was collected and dried in a speed vac. As a control, celllysate was also incubated with N3′-ethyl biotin alkyne 118 or biotinalkyne without Cu(I) catalyst. Another control is using untreated celllysate to perform the same isolation procedure.

As shown in FIG. 6, both control experiments without Cu(I) catalyst, andwithout Ac₄GlcNAz treatment, show very low background using N3′-ethylbiotin alkyne 118, indicating the azido containing glycoproteins wereselectively isolated by labeling with N3′-ethyl biotin alkyne 118 viaformation of triazole linkage, followed by capture with streptavidinagarose via interaction of N3′-ethyl biotin with streptavidin. One majorproblem using biotin alkyne is the isolated proteins are contaminatedwith streptavidin (SA), which is leached out from agarose beads by theharsh, denaturing condition. These results demonstrate using N3′-ethylbiotin has great advantage in gentle release to isolate clean proteinsfor downstream analysis.

Example 23: Cell Separation Using Strepavidin-N3′-Ethyl Biotin System

Applicants have identified certain biotin analogs which show optimalreversible interaction with streptavidin, that allows (1) fast andcomplete binding and (2) fast and complete release, under mild,physiological conditions. One of biotin analogs identified, namedN3′-ethyl biotin (k_(D)˜0.8 nM), has fast on-rate (k_(on)˜1.5×10⁵M⁻¹S⁻¹), and also fast off-rate (koff˜5.8×10⁻² S⁻¹) in the presence of 1mM bis-biotin competing reagent. Instead, biotin is almost irreversibleunder the same condition. These reversible interactions betweenN3′-ethyl biotin and streptavidin provide a unique method for reversibleimmobilization of N3′-ethyl biotinylated antibody on magneticstreptavidin beads for cell separation applications.

In order to evaluate cell separation efficiency usingstreptavidin-N3′-ethyl biotin system, the N3′-ethyl biotin 17 (shownbelow) was first conjugated with mouse anti-human CDx monoclonalantibodies (mAb). The mAbs were also labeled with AlexaFluor 488 (AF488)to allow for flow cytometry analysis.

General Procedure of Antibody Labeling with N3′-Ethyl Biotin.

A solution of antibody (both primary and secondary antibody) wasprepared at ˜3 mg/mL in 0.1 M sodium bicarbonate. N3′-ethyl biotinsuccinimidyl ester 17 was dissolved in DMSO at ˜20 mg/mL. A molar ratioof 10, 20, and 30 equivalents of N3′-ethyl biotin succinimidyl ester 17was added to the protein solution with stirring (magnetic) atatmosphere, respectively. The reaction mixture was incubated at roomtemperature for one hour. The N3′-ethyl biotin-antibody conjugate waspurified by spin column using BIO-RAD P-30 resin equilibrated withphosphate-buffer saline (PBS).

General Procedure of Antibody Labeling with AlexaFluor Dyes.

A solution of antibody (both primary and secondary antibody) wasprepared at ˜3 mg/mL in 0.1 M sodium bicarbonate. Alexa Fluor® dyesuccinimidyl ester was dissolved in DMSO at ˜10 mg/mL. A molar ratio of20 equivalents of Alexa Fluor® 488 succinimidyl ester (LifeTechnologies, San Diego, Calif., SKU # A20000), while for Alexa Fluor®647 succinimidyl ester (Life Technologies, San Diego, Calif., SKU #A20006) a molar ratio of 8 was used. The succinimidyl esater was addedto the protein solution with stirring (magnetic) at atmosphere. Thereaction mixture was incubated at room temperature for one hour. TheAlexa Fluor® dye-antibody conjugate was purified by spin column usingBIO-RAD® P-30 resin equilibrated with phosphate-buffer saline (PBS).

Measurement of Degree of Labeling. Determination of Degree of Labeling(DOL) of N3′-Ethyl Biotin.

The determination of accessible biotin analog in the antibody conjugateswas tested using the commercial available FluoReporter® BiotinQuantitation Assay Kit (Life Technologies, San Diego, Calif., SKU #F30755). Briefly, 2 mL of HABA/avidin complex was added into twoseparate test tubes. To each tube, 100 μL of PBS and N3′-ethylbiotin-antibody conjugate were added, respectively, and incubated for 10min with gentle stirring. The absorbance of both samples was measured at500 nm, and calculated the difference in absorbance between the twosamples (ABS=Blank ABS−Sample ABS). The degree of labeling (DOL) ofbiotin analogs is calculated using following equation:

DOL=(ABS×BSF×Mw protein×1000)/(C×0.05)

BSF: conversion factor (2.37×10⁻⁸); C: concentration of protein (mg/mL).

Determination of Degree of Labeling (DOL) of AF488 and AF647.

A simple method for estimating the degree of labeling (DOL) isdetermined by direct measuring the protein absorbance at 280 nm andAF488 dye (AF647 dye) absorbance at the absorption maximum. The proteinconcentration is calculated as follows:

${{Protein}\mspace{14mu} {concentration}\mspace{14mu} (M)} = \frac{A_{280} - {XA}_{dye}}{ɛ_{protein}}$

where the molar extinction coefficient (E) of a typical IgG at 280 nm is203,000 cm⁻¹M⁻¹. The molar extinction coefficient (E) of AF488 dye at494 nm is 71,000 cm⁻¹M⁻¹, and the correction factor (X) for AF488 dye is0.11. The molar extinction coefficient (E) of AF647 dye at 649 nm is240,000 cm⁻¹M⁻¹, and the correction factor (X) for AF647 dye is 0.03.The degree of labeling (DOL) is calculated as follows:

${DOL} = \frac{A_{dye}ɛ_{protein}}{\left( {A_{280} - {XA}_{dye}} \right)ɛ_{dye}}$

Preparation of Cells.

The cell separation was evaluated with a cell model system of humanperipheral T lymphocytes (CD3⁺, CD4⁺, and CD8⁺ cells).

The human peripheral blood mononuclear cells (PBMCs) were prepared byNH₄Cl lysis from human peripheral blood obtained from healthy volunteerdonors. NH₄Cl lysis buffer (150 mM NH₄Cl, 10 mM NaHCO₃ and 1 mM EDTA)was prewarmed in 37° C. water bath. Each 5 mL whole blood was added with45 mL NH₄Cl lysis buffer in a 50 mL centrifuge tube, and was agitatedfor 15 min at room temperature. The sample was centrifuged at 350×g for5 min, and supernatant was removed by pipette. The cell pellet wasresuspended in 15 mL isolation buffer (DPBS, 0.1% BSA, 2 mM EDTA), andcentrifuged again at 350×g for 5 min. Supernatant was removed bypipette, and the cell pellet was resuspended in 2 mL isolation buffer.Count cells and adjust cell concentration to 3×10⁷ cells/mL.

General Procedure of Cell Separation.

Briefly, PBMCs (3×10⁷ cells) were first incubated with mouse anti-humanCDx (CD3, CD4, and CD8) mAb-N3′-ethyl biotin 17 conjugate on ice for 10min, respectively. Then streptavidin M280 Dynabeads® magnetic beads(Life Technologies, San Diego, Calif., SKU #112-05D) were added andincubated for 10 min under rolling and tilting at room temperature. Thecell mixture was placed on a magnet for 1 min, and the supernatant wasremoved. The magnetic labelled cells were washed, and then the cellswere released from magnetic beads by incubating with 1 mM bis-biotin for5 min. The amount and viability of isolated cells were measured byCountess® automated cell counter (Life Technologies, San Diego, Calif.,SKU # C10227). The depletion efficiency and purity of cells wereanalysed by flow cytometry. The results are summarized in FIGS. 7A-7Cand the table below. By demonstrating the feasibility of cell separationbased on three different cell surface markers, a simple work-flow (twostep incubations, one step wash, and one step detachment, FIG. 7A hasbeen developed. The whole cell separation process can be done in lessthan 30 min to achieve high recovery (>80%), high purity (>92%), andhigh viability (>92%). By using streptavidin M280 Dynalbeads, eachtarget cell was captured by multiple magnetic beads (FIG. 7B) throughinteractions between N3′-ethyl biotin and streptavidin, and can beeasily separated by a simple magnet (FIG. 7B) to achieve high depletionefficiency (98-100%, FIG. 7C). N3′-Ethyl biotinylated mAb was fast andcomplete released from the streptavidin magnetic beads using 1 mMbis-biotin under physiological conditions to give high recovery, highpurity, and high viability of cells (FIG. 7C).

Recovery Purity, and Viability of Isolated Cells

Cell Method type Recovery^([a]) Purity^([b]) Viability^([a]) Primary CD387 ± 6% 96 ± 2% 96 ± 2% Ab Primary CD4 84 ± 8% 92 ± 2% 92 ± 3% AbPrimary CD8 72 ± 5% 93 ± 3% 93 ± 2% Ab Secondary CD3 92 ± 4% 96 ± 2% 97± 2% Ab Secondary CD4 88 ± 6% 97 ± 2% 94 ± 2% Ab Secondary CD8 82 ± 5%96 ± 2% 95 ± 3% Ab ^([a])Each recovery and viability is reported themean of independent triplicate experiments based on Countess ®measurement. ^([b])Each purity is reported the mean of independenttriplicate experiments based on flow cytometry measurement. Error barsrepresent standard deviation of independent triplicate experiments.

Method One:

To a 1 mL PBMCs (˜3×10⁷ cells) in a 15 mL centrifuge tube was added 2 μLmouse anti-human CDx mAb-N3′-ethyl biotin 17 conjugate (1 mg/mL in PBS),and incubated on ice for 10 min. Then, 10 mL isolation buffer was added,and centrifuged at 350×g for 5 min. 8 mL Supernatant was carefullyremoved and discarded by pipette, and 70 μL streptavidin M280 Dynabeads®magnetic beads (15 mg/mL) were added and incubated for 10 min at roomtemperature under rolling and tilting. The tube was placed on a magnetfor 1 min, and supernatant was removed. Then, the tube was removed fromthe magnet, and added 5 mL isolation buffer and resuspended thebead-bound cells by gentle pipetting 5 times. The tube was placed on amagnet for 1 min, and supernatant was removed. The wash step wasrepeated twice. After three washes, 1 mL release buffer (1 mM bis-biotinin isolation buffer) was added, and incubated for 5 min at roomtemperature under rolling and tilting. The suspension was mixed bypipetting 10 times, and the tube was placed on a magnet for 1 min. Thesupernatant containing the bead-free cells was transferred to a newtube, and centrifuged at 350×g for 5 min. The supernatant was removedand discarded, and the cell pellet was suspended in 1 mL isolationbuffer.

Method Two:

To a 1 mL PBMCs (˜3×10⁷ cells) in a 15 mL centrifuge tube was added 2 μLmouse anti-human CDx mAb (1 mg/mL in PBS), and incubated on ice for 10min. Then, 10 mL isolation buffer was added, and centrifuged at 350×gfor 5 min. 8 mL Supernatant was carefully removed and discarded bypipette, and 70 μL goat anti-mouse (GAM) IgG-N3′-ethyl biotin 17conjugate coated streptavidin M280 Dynabeads® magnetic beads (preparedby incubation 10 g GAM IgG-N3′-ethyl biotin conjugate 17 per mg ofstreptavidin M280 Dynabeads® magnetic beads, 15 mg/mL) was added andincubated for 10 min at room temperature under rolling and tilting. Thetube was placed on a magnet for 1 min, and supernatant was removed.Then, the tube was removed from the magnet, and added 5 mL isolationbuffer and resuspended the bead-bound cells by gentle pipetting 5 times.The tube was placed on a magnet for 1 min, and supernatant was removed.The wash step was repeated twice. After three washes, 1 mL releasebuffer (1 mM bis-biotin in isolation buffer) was added, and incubatedfor 5 min at room temperature under rolling and tilting. The suspensionwas mixed by pipetting 10 times, and the tube was placed on a magnet for1 min. The supernatant containing the bead-free cells was transferred toa new tube, and centrifuged at 350×g for 5 min. The supernatant wasremoved and discarded, and the cell pellet was suspended in 1 mLisolation buffer.

General Procedure of Cell Counting and Viability Testing by Countess®Automated Cell Counter.

10 μL of cell sample and 10 μL trypan blue stain solution were mixed bypipette. 10 μL of the sample mixture was added to the chamber ports onone side of the Countess® cell counting chamber slide. Then, theCountess® cell counting chamber slide was inserted into the slide inleton the instrument. The cell concentration and viability weresimultaneously recorded by the instrument.

General Procedure of Cell Analysis by Flow Cytometry.

The cell analysis was performed using a BD® LSR II Flow Cytometry(Becton Dickinson, Franklin Lakers, NJ). The blank PBMCs were used as acontrol sample to set up forward angle light scatter (FSC), side anglelight scatter (SSC), and photomultiplier tube (PMT) voltage to get nicepopulation of lymphocytes, monocytes, and granulocytes. The sameinstrument settings were used to run the antibody labeled PBMCs,collecting 20,000 events for each sample. The AF488 signals werecollected using an excitation laser at 488 nm and an emission filter at515-545 nm. The AF647 signals were collected using an excitation laserat 633 nm and an emission filter at 650-670 nm.

Example 24: Cell Separation Based on Dual Cell Surface Markers UsingBiotin and Biotin Analog with Different Binding Affinity

Human peripheral T lymphocytes based on dual cell surface markers (CD3⁺and CD4⁺) were chosen to demonstrate cell separation. Anti-CD4 mAb waslabelled with the biotin molecule and AlexaFluor 647 (AF647) (for flowcytometry analysis purpose); and labelled anti-CD3 mAb with N3′-ethylbiotin 17 and AlexaFluor 488 (AF488). PBMCs (3×10⁷ cells) were incubatedwith anti-CD4 mAb-biotin conjugate and anti-CD3 mAb-N3′-ethyl biotin 17conjugate simultaneously on ice for 10 min. Then streptavidin M280Dynabeads® magnetic beads were added and incubated for 10 min at roomtemperature. After washing step to remove unbound reagents, the magneticlabeled cells were selectively released by incubating with 1 mMbis-biotin for 5 min to yield beads-free CD3+CD4⁻ cells.

As shown in FIG. 8, after incubation with biotinylated anti-CD4 mAb andN3′-ethyl biotinylated anti-CD3 mAb, the cells either CD3⁺ or CD4⁺ havebeen captured by streptavidin M280 Dynabeads® magnetic beads withdepletion efficiency (˜100%). After incubating with 1 mM bis-biotin,CD3+CD4⁻ cells were selectively detached from magnetic beads with 80±5%recovery, 92±3% purity and 95±2% viability, and CD4⁺ cells remain boundto magnetic beads. The results demonstrate that the N3′-ethyl biotin canbe selectively detached from streptavidin without interferingbiotin-streptavidin interactions by control of release condition. Thisprovides a simple one-step cell selection method based on dual cellsurface markers.

Although the disclosed teachings have been described with reference tovarious applications, methods, and compositions, it will be appreciatedthat various changes and modifications may be made without departingfrom the teachings herein. The foregoing examples are provided to betterillustrate the present teachings and are not intended to limit the scopeof the teachings herein. Certain aspects of the present teachings may befurther understood in light of the following claims.

1. A biotin derivative having structural formula (I):

wherein: X is S or O; R₁ is selected from H and a derivative group; R₂is selected from H and a derivative group; Y is O or absent; L is absentor is a linker; R₃ is selected from —OR₄, —COOR₄, a reactive group, aprotein, a peptide, an amino acid, a dextran, a monosaccharide, adisaccharide, a trisaccharide, an oligosaccharide, a polysaccharide, ahormone, a lipopolysaccharide, a nucleotide, an oligonucleotide, a smallmolecule, a cell, a microplate, and a microparticle; R₄ is selected fromH and a derivative group; wherein at least one of R₁ and R₂ is H;wherein if X is O, at least one of R₁ and R₂ is not H, or Y is O; orstructural formula (II):

wherein: X is S or O; R is selected from H and a derivative group; R₂ isselected from H and a derivative group; L is absent or is a linker; R₃is selected from —OR₄, —COOR₄, a reactive group, a protein, a peptide,an amino acid, a dextran, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a hormone, alipopolysaccharide, a nucleotide, an oligonucleotide, a small molecule,a cell, a microplate, and a microparticle; R₄ is selected from H and aderivative group; wherein at least one of R₁ and R₂ is H. 2-76.(canceled)
 77. A composition comprising a biotin derivative of claim 1and a solvent.
 78. (canceled)
 79. A method of immobilizing a biotinderivative to a solid support, comprising contacting a solid supportwith a biotin derivative of claim 1, wherein the solid support comprisesa biotin-binding moiety. 80-107. (canceled)
 108. A method of detecting aprotein in a sample, comprising the steps of: a. providing a biotinderivative of claim 1, wherein R₃ is a reactive group; b. providing asample comprising a protein comprising a complementary group capable ofreacting with the reactive group; c. providing a biotin-binding moietycomprising a detectable moiety; d. contacting the biotin derivative withthe sample to provide a labeling mixture; e. contacting the labelingmixture with the biotin-binding moiety; and f. detecting the protein.109-114. (canceled)
 115. A method of detecting a protein in a sample,comprising the steps of: a. providing a biotin derivative of claim 1,wherein R₃ is a reactive group; and wherein the linker is present andcomprises a detectable moiety. b. providing a sample comprising aprotein comprising a complementary group capable of reacting with thereactive group; c. contacting the biotin derivative with the sample; andd. detecting the protein. 116-120. (canceled)
 121. A method of isolatinga protein in a sample, comprising the steps of: a. providing a biotinderivative of claim 1, wherein R₃ is a reactive group; b. providing asample comprising a protein comprising a complementary group capable ofreacting with the reactive group; c. providing a solid supportcomprising a biotin-binding moiety; d. contacting the biotin derivativewith the sample to provide a labeling mixture; e. contacting thelabeling mixture with the solid support; and f. allowing the protein tobind to the solid support to provide the isolated protein. 122-126.(canceled)
 127. A method of isolating a cell in a sample, comprising thesteps of: a. providing a sample comprising at least one cell; b.providing a biotin derivative of claim 1, wherein R₃ is a reactivegroup; c. providing an antibody comprising a complementary group capableof reacting with the reactive group and capable of binding to a proteinon the cell; d. providing a solid support comprising a biotin-bindingmoiety; e. contacting the biotin derivative with the antibody to providea biotin-labeled antibody; f. contacting the biotin-labeled antibodywith the sample to provide a labeling mixture; g. contacting thelabeling mixture with the solid support; and h. allowing the cell tobind to the solid support to provide the isolated cell. 128-132.(canceled)
 133. A method of labeling a protein, comprising the steps of:a. providing a biotin derivative of claim 1, wherein R₃ is a reactivegroup; b. providing a sample comprising a protein comprising acomplementary group capable of reacting with the reactive group; and c.contacting the biotin derivative with the sample to provide a biotinderivative-labeled protein. 134-137. (canceled)
 138. The method of claim133, further comprising the steps of: a. providing a biotin-bindingmoiety comprising a detectable moiety; and b. contacting thebiotin-binding moiety with the biotin derivative-labeled protein. 139.The method of claim 138, wherein the biotin-binding moiety is selectedfrom an avidin or a strepavidin.
 140. The method of claim 138, whereinthe detectable moiety is a chromophore, a fluorescent dye, a fluorescentprotein, a nanocrystal, an enzyme, a molecule comprising an atom whichis a radioisotope, or an atom which is a radioisotope.
 141. A method oflabeling a protein in a sample, comprising the steps of: a. providing abiotin derivative of claim 1, wherein R₃ is a reactive group; andwherein the linker is present and comprises a detectable moiety; b.providing a sample comprising a protein comprising a complementary groupcapable of reacting with the reactive group; and c. contacting thebiotin derivative with the sample to provide a labeled protein.
 142. Themethod of claim 141, wherein the reactive group is an alkyne, and thecomplementary group is an azide.
 143. (canceled)
 144. The method ofclaim 141, wherein the reactive group is an azide and the complementarygroup is an alkyne.
 145. (canceled)
 146. The method of claim 141,wherein the detectable moiety is a chromophore, a fluorescent dye, afluorescent protein, a nanocrystal, an enzyme, a molecule comprising anatom which is a radioisotope, or an atom which is a radioisotope.
 147. Amethod of labeling a biotin derivative comprising the steps of: a.providing a biotin derivative of claim 1; b. providing a biotin-bindingmoiety; and c. contacting the biotin derivative with the biotin-bindingmoiety to provide the labeled biotin derivative. 148-149. (canceled)150. A kit comprising a biotin derivative of claim 1 and a biotinbinding moiety.
 151. The kit of claim 150, wherein the biotin-bindingmoiety is selected from an avidin or a strepavidin.
 152. The kit ofclaim 150, wherein the biotin-binding moiety comprises a detectablemoiety.
 153. The kit of claim 149, wherein the detectable moiety is achromophore, a fluorescent dye, a fluorescent protein, a nanocrystal, anenzyme, or a radioisotope.